1. Field of the Invention
The present invention relates to a method for calibrating an instrument used to analyze particles, and more particularly, concerns a method and materials for calibrating flow cytometry instruments prior to using such instruments for obtaining at least one light-related signal from particles passing through the instrument.
2. Background Description
There are a number of commercially available instruments used for particle or cellular analysis. Characteristics of particles, particularly biological particles or cells, may be determined by techniques in which the particles remain relatively fixed while being analyzed or while the particles are moving in a stream or carried in a suspension. Flow cytometers are known and available for analyzing or detecting certain characteristics of particles which are in motion. In typical flow cytometry instruments, cells or other biological particles are caused to flow in a liquid stream so that each particle, preferably one at a time, passes through a sensing region which measures physical or chemical characteristics of the particles. Although a variety of signals may be detected for association with different characteristics of the particles, including electrical, acoustical and radioactive, flow cytometers commonly rely on optical signals for the analysis of particles passing through the instrument. In any event, whether the analysis instrument is used to analyze particles in a static or dynamic state, preliminary set-up steps are normally required in preparing the instrument for use. Insofar as cells or other biological particles are very small and the signals to be detected with respect to those particles are often at a low magnitude, proper calibration of the instrument, prior to conducting the analysis, is desirable to assure reliable test results.
Current techniques for calibrating instruments useful for biological particle or cellular analysis are highly operator dependent. In other words, the operator's judgment and experience is called upon during the calibration procedures for adjusting the controls, establishing proper thresholds and maintaining a set of standards for the particular tests to be conducted. Indeed, while experienced operators are quite capable of properly calibrating an instrument prior to use, much guesswork and speculation is used by such an operator to sense that proper calibration has been attained. For instance, in aligning various optical elements of an instrument, such as a flow cytometer, the person doing the calibration typically views a screen or a CRT and, by eyeball judgment, in conjunction with adjustment of various controls, determines that the instrument has been calibrated. Of course, in employing the typical eyeball calibration techniques, reproducibility of test results could be compromised.
Flow cytometers and other biological particle analysis instruments are normally calibrated with particles which simulate or approximate the types of particles or cells which are expected to undergo actual analysis. Thus, calibration particles should be selected so that they have characteristics similar to the particles to be tested for, such as size, volume, surface characteristics, granularity properties, color features (cellular stains, dyes, immunofluorescent tags and the like). Past and current calibration procedures for flow cytometry instruments include the utilization of chicken red blood cells for the calibration steps. While chicken red blood cells are reliable for some aspects of the calibration procedures, they, nevertheless, are not entirely satisfactory, particularly because of spectral deficiencies in some light-related characteristics. In addition to biological samples for calibration purposes, microspheric beads have become available for calibrating cellular analysis instruments. For example, beads made out of latex material for use in calibrating a particle counting instrument were described in U.S. Pat. No. 4,331,862. Calibration beads specifically directed for calibrating flow cytometry instruments are commercially available from the Flow Cytometry Standards Corporation, Research Triangle Park, N.C.
Even though the availability of calibration beads has led to improvements in the calibration techniques of particle analysis instruments there is still a substantial amount of operator guesswork in determining when the instrument has been properly calibrated. Accordingly, there is still a need and desire to establish calibration procedures which not only provide reproducible test results, but also eliminate the speculation and guesswork attendant to currently utilized calibration techniques. It is toward the fulfillment of such improvements that the present invention is directed.